RNA binding proteins (RBPs) exert an especially strong effect on gene regulation in kinetoplastids compared to other organisms since kinetoplastids do not regulate RNA polymerase II transcription and instead rely on posttranscriptional gene regulatory mechanisms. Recent proteomic studies revealed that many T. brucei RBPs are subject to posttranslational modifications (PTMs) such as serine/threonine phosphorylation and arginine methylation. In other systems, PTMs expand RBP function and contribute to their regulation; however, almost nothing is known about the mechanisms by which PTMs impact the functions of kinetoplastid RBPs. ZFP3 is a regulatory RBP that is essential in mammalian bloodstream form (BF) T. brucei and stimulates differentiation from the BF to the insect procyclic form (PF). ZFP3 is, thus, critical for T. brucei survival and pathogenesis. The multifunctional ZFP3 binds and stabilizes dozens of mRNAs, stimulates translation of EP1 procyclin mRNA through PF-specific ribosome association, and is recruited to cytoplasmic mRNP granules in PF during starvation stress. Proteomic analyses from our lab and others showed that the 14 kDa ZFP3 contains two methylarginine and two phosphoserine marks. Here, we propose to test the hypothesis that PTMs regulate and diversify ZFP3 functions, thereby contributing to its critical roles in BF and PF T. brucei. Our preliminary data indicate that arginine methylation is essential for the morphological manifestation of ZFP3 action in PF termed ?nozzle?. In Aim 1, we will compare cells that overexpress epitope tagged wild type (WT) ZFP3 to those overexpressing hypomethylated, hypophosphorylated, methylmimic, or phosphomimic ZFP mutants. We will measure the capacity of ZFP3 and its PTM variants to potentiate BF to PF differentiation, bind and modulate the stabilities of specific mRNAs, stimulate EP1 procyclin translation, and regulate ZFP3 association with ribosomes, stress granules, and other binding partners. We will also perform RNAseq and RIPseq studies to define the global impacts of PTMs on ZFP3 function. In Aim 2, we will quantify classes of PTMs on ZFP3 in BF and PF towards a comprehensive understanding of this protein's posttranslational regulation during the life cycle. Using novel, label-free mass spectrometry approaches we will determine the fraction of ZFP3 harboring methylarginine phosphoserine/threonine/tyrosine, methyllysine, and acetylysine, and we will define differences between BF and PF parasites. We will examine the capacity of specific PTMs to affect each others' deposition, leading to ZFP molecules harboring distinct PTM patterns (?PTM crosstalk?) using a range of mass spectrometry approaches, including top-down analysis of intact ZFP3 molecules. Collectively, the proposed studies will provide insight into the mechanisms by which PTMs diversify and modulate the functions of a key trypanosome regulatory RBP. They will also provide the first insights into PTM crosstalk in trypanosomes and provide a methodological framework for similar analyses of other critical trypanosome RBPs.